A novel electrophoretic technique designed to modify the ratio of magnesium isotopes inside the creatine kinase active sites. A preliminary report

Abstract

A simple and efficient preparative electrophoretic technique has been proposed to obtain a modified creatine kinase (CK, E.C.2.7.3.2) molecule with an increased content of ²⁵Mg in the active site. A key point of the method is the special design of a 0.9 × 12.0 cm column for ascendent electrophoresis, packed consecutively, from the bottom to the top, with layers of 30 % PAAG (polyacrylamide grade), ²⁵Mg²⁺-containing 7.5 % PAAG, enzyme-binding ADP Sepharose and 2.2 % agarose gels, based on different tris–glycine and tris–HCl separation buffer systems. The isotope substitution process was a result of simultaneous desorption of enzyme from ADP Sepharose and electrically directed extensive flow of ²⁵Mg²⁺ cations through the porous gel matrix. Greater than 8-fold ²⁵Mg enrichment, i.e. a 10.2–86.3 % increase of ²⁵Mg contribution to total enzyme magnesium, has been reached. The modified ²⁵Mg-rich CK samples manifest higher (2.4-fold increase) values of specific catalytic activity when compared with intact (control) ones.

Keywords:

• Creatine kinase
• Isotope effect
• Isotope enrichment
• Magnesium-25
• Spin-selective process

Acknowledgements

This work was partly supported by La Sapienza Biomedica Foundation, Varese, Italy. We thank the Voting Member of Russian Academy of Sciences, Prof. Anatoly L. Buchachenko, and Prof. Dr Alexandre V. Shishkov (N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia) for the stimulating discussions of the data presented.